1. Field of the Invention:
This invention relates to novel maltooligosaccharide derivatives and to reagents for the determination of .alpha.-amylase activity wherein the maltooligosaccharide derivatives are used as substrates.
2. Prior Art Statement
Various diseases are diagnosed by determining the activity of .alpha.-amylase contained in body fluids such as pancreatic juice, urine, etc. For the determination of the activity of .alpha.-amylase, for example, a method that utilizes maltooligosaccharides such as maltotetraose, maltopentaose, maltohexaose, etc. as substrates is known. According to this method, a maltooliosaccharide and .alpha.-glucosidase are acted upon samples containing .alpha.-amylase to liberate glucose from the substrate, and the quantities of the glucose thus liberated are measured, whereby the activity values of .alpha.-amylase are found.
The glucose so produced is determined, for example, by a quantitative measurement method that utilizes a glucoseoxidase/peroxidase/pigment series, another measurement method that utilizes a hexokinase/phosphoglucomutase/glucose-6-phosphate dehydrogenase/NADH series, etc. Since .alpha.-glucosidase is difficult to act on tetrasaccharides or higher oligosaccharides such as maltopentaose and acts satisfactorily on trisaccharides or lower oligosaccharides such as maltose, maltotriose, etc., the activity of .alpha.-amylase can be determined by using the aforementioned substrate and measuring the glucose. However, though slightly, .alpha.-glucosidase acts on maltopentaose as substrate and because of this, elevated blank values are measured, which leads to larger errors in measurements. Owing to the decomposition action of .alpha.-glucosidase to the substrate, it is not preferred to prepare a one-part liquid reagent containing both .alpha.-glucosidase and the substrate because its reagent stability is impaired
Another determination method of the activity is proposed, which uses substrates wherein a phenyl group, a naphthyl group or their derivatives are bonded to the reducing ends of maltooligosaccharides as a glycon Examples of such substrates include p-nitrophenylmaltopentaoside (Japanese Patent Publication No. 57-53079 (1982)), p-nitrophenylmaltohexaoside (Japanese Patent Publication No. 57-53079 (1982)), p-nitrophenylmaltoheptaoside (Japanese Patent Unexamined Publication No. 54-51892 (1979)), 2,4-dichlorophenylmaltopentaoside (Japanese Patent Unexamined Publication No. 56-35998 (1981)),etc. When these substrates are used, aglycons are liberated, and optical measurement of the liberated aglycons, e.g. p-nitrophenol permits easy determination of the activity of .alpha.-amylase
This method also has the defect that .alpha.-glucosidase acts on the substrates, though slightly, and consequently, elevated blank values are measured Because of the substrate-decomposing action of .alpha.-glucosidase, it is difficult to prepare a onepart liquid type reagent containing at once .alpha.-glucosidase and the substrates, as in the case of the foregoing method of determining glucose.
In order to remove the drawbacks, there is provided a method of employing substrates of such type that the hydroxyl group at the 6-position of the non-reducing terminal glucose of maltooligosaccharides is modified For instance, Japanese Patent Unexamined Publication No. 60-237998 (1985) discloses maltooligosides as substrates wherein OH group at 6-position of the non-reducing terminal glucose of maltooligosaccharides is substituted, for example, by a halogen atom, --OR, --OCOR, --OSO.sub.2 R or --NHR (wherein R is alkyl, phenyl, pyridyl radicals, etc.) and the reducing terminal glucose of it is bonded with a substituted or non-substituted phenyl group as aglycon. The OH group at the 6-position of the non-reducing terminal glucose being substituted, the substrates do not undergo any decomposition by means of .alpha.-glucosidase.
However, such substrates having a substituent introduced solely at the 6-position are difficult to synthesize and are produced only in low yields.
Japanese Patent Unexamined Publication No. 60-54395 (1985) discloses maltooligosides for use as substrates, wherein 0H groups at the 4- and 6-positions of the non-reducing terminal glucose of maltooligosaccharides are substituted by an alkyl group, an alkoyl group or a phenyl group and the reducing terminal glucose of it is bonded with aglycon. Because of the fact that OH groups at the 4-position and 6-position of the nonreducing terminal glucose are substituted, the substrates are little susceptible to being decomposed by .alpha.-glucosidase The substrates are, however, poorly water-soluble, having two OH groups substituted, and hence it is impossible to prepare a high concentration solution Therefore, it is disadvantageous in that the substrates cannot be dissolved to a sufficient concentration for the determination of c-amylase activity.